Chelating agents

ABSTRACT

The invention provides a complexant compound of formula I 
     
       
         R 3 S(CR 1   2 ) n N(R 2 ) i (CR 1   2 ) n X(CR 1   2 ) n N(R 2 ) i (CR 1   2 ) n SR 3   (I)  
       
     
     (wherein 
     each n, which may be the same or different, is an integer 2, 3 or 4 (preferably 2); 
     each i, which may be the same or different, represents 0 or 1; 
     each R 3 , which may be the same or different, is H or a thiol protecting group, preferably a protecting group; 
     X is O, S, N, NR 4  or a substituted phosphorus (eg. oxo substituted phosphorus), preferably S or N; 
     each R 4 , which may be the same or different, is hydrogen or an optionally substituted organic group; 
     each R 1 , which may be the same or different, is hydrogen or an optionally substituted organic group, or a moiety CR 1   2  may represents a carbonyl group or two, three or four R 1 s on two different carbons together with those carbons and any intervening atoms may represent an optionally substituted saturated or unsaturated homocyclic or heterocyclic ring; and preferably, at least one CR 1   2  moiety is other than CH 2  or CH(CH 3 )) or a salt or complex thereof, wherein optionally at least one of the R 1 , R 2 , R 3  and R 4  moieties is coupled directly or indirectly to a vector moiety.

This application is a divisional application of allowed U.S. applicationSer. No. 08/833, 995, filed Apr. 11, 1997, now U.S. Pat. No. 6,007,529.

This invention relates to complexants and metallated complexes thereofand to their use in diagnostic, therapeutic and prophylacticcompositions, in particular to the use of such complexants metallatedwith radionuclides as diagnostic imaging and therapeutic agents.

Radiopharmaceuticals, the class of drug compounds containingradionuclides, are useful for the diagnosis and treatment of variousdisease states, in particular certain cancers.

The radionuclide in such radiopharmaceuticals may be a metal (eg. atransition metal or lanthanide) or a nonmetal (eg. an iodine or hydrogenradionuclide). Where the radionuclide is a metal, it is conventionallyadministered as a complex (usually a chelate complex) of a mono- orpolyatomic ion of or containing the metal, with a complexing agent. Thepresent invention is particularly concerned with complexed metalradionuclides and complexants which can be metallated with metal ionradionuclides.

In the use of complexed metal radiopharmaceuticals, the diagnostic ortherapeutic properties are selected by appropriate selection of themetal radionuclide (eg. by virtue of its decay pattern or half life)while the biodistribution and bioelimination properties are selected byappropriate selection of the complexant and, if desired of a vectormoiety coupled directly or indirectly to the complexant so as to causethe complexed radionuclide to be targeted to a particular body site ortissue type, eg. cancerous tissue.

Examples of complexants that have been proposed for use with metalradionuclides in therapeutic or diagnostic compositions include theterpyridine chelants disclosed in WO (TMT cases) and the BAT chelantsdiscussed by Ohmomo et al. in J. Med. Chem. 35: 157-162 (1992) and byKung et al. in J. Nucl. Med. 25: 326-332 (1984).

Nevertheless there is a continuing need for complexants which arecapable of adequately complexing diagnostic and therapeutic metalradionuclides and which preferably also may be coupled to effectivevector moieties so as to target the complexed radionuclide to a desiredtarget site within the patient's body.

In particular there is a continuing need for complexants that may beused to complex both diagnostically effective metal radionuclides andtherapeutically effective metal radionuclides. In this way a diseasesite may be imaged and treated using diagnostic and therapeutic agentswhich have substantially identical biodistributions since the carrierportion of the metal: carrier complex, which determines thebiodistribution pattern of the complex, may be the same in both thediagnostic agent and the therapeutic agent.

We have now found that a new class of complexants possesses appropriateproperties in this regard.

The novel complexants are referred to as N₂S₂X complexants since theycontain a carbon chain interrupted, in order by S, N, X, N and Sheteroatoms (where X is an O, S, N or P heteroatom). Between theseheteroatoms there are carbon chains 2, 3 or 4 atoms long. Suchcomplexants, and the salts and complexes thereof, including the targetedcomplexes thereof, form one aspect of the invention.

Viewed from a further aspect the invention provides a complexantcompound of formula I

R³S(CR¹ ₂)_(n)N(R²)_(i)(CR¹ ₂)_(n)X(CR¹ ₂)_(n)N(R²)_(i)(CR¹₂)_(n)SR³  (I)

(wherein

each n, which may be the same or different, is an integer 2, 3 or 4(preferably 2);

each i, which may be the same or different, represents 0 or 1;

each R³, which may be the same or different, is H or a thiol protectinggroup, preferably a protecting group;

X is 0, S, N, NR⁴ or a substituted phosphorus (eg. oxo substitutedphosphorus), preferably S or N;

each R⁴, which may be the same or different, is hydrogen or anoptionally substituted organic group;

each R¹, which may be the same or different, is hydrogen or anoptionally substituted organic group, or a moiety CR¹ ₂ may represents acarbonyl group or two, three or four R¹s on two different carbonstogether with those carbons and any intervening atoms may represent anoptionally substituted saturated or unsaturated homocyclic orheterocyclic ring; and preferably, at least one CR¹ ₂ moiety is otherthan CH₂ or CH(CH₃)) or a salt or complex thereof, wherein optionally atleast one of the R¹, R², R³ and R⁴ moieties is coupled directly orindirectly to a vector moiety.

Viewed from a further aspect the invention provides a pharmaceuticalcomposition comprising an effective amount (eg. an amount effective toenhance image contrast in in vivo imaging or an amount sufficient toachieve a desired therapeutic effect) of a complex of an optionallyvector coupled complexant of formula I together with at least onepharmaceutically effective carrier or excipient.

Viewed from a still further aspect the invention provides the use of acomplex of an optionally vector coupled complexant of formula I for themanufacture of a contrast medium for use in a method of diagnosisinvolving administration of said contrast medium to an animate subjectand generation of an image of at least part of said subject.

Viewed from a still further aspect the invention provides the use of acomplex of an optionally vector coupled complexant of formula I for themanufacture of a therapeutic agent, eg. a radiopharmaceutical, forexample for use in tumor therapy.

Viewed from a still further aspect the invention provides a method ofgenerating an image of an animate human or non-human (preferablymammalian or avian) animal subject involving administering a contrastagent to said subject, eg. into the vascular system or the gi tract, andgenerating an image of at least a part of said subject to which saidcontrast agent has distributed, eg. by X-ray, MR, ultrasound,scintigraphic, PET, SPECT, electrical impedance, light or magnetometricimaging modalities, characterised in that as said contrast agent is useda complex of an optionally vector coupled complexant of formula I.

Viewed from a still further aspect the invention provides a method oftreatment of an animate human or non-human (preferably mammalian oravian) animal subject involving administering a therapeutic agent tosaid subject, eg. into the vascular system or the gi tract,characterised in that as said therapeutic agent is used a complex of anoptionally vector coupled complexant of formula I.

Viewed from a yet further aspect the invention provides a process forthe preparation of a complex of an optionally vector coupled complexantof formula I, said process comprising metallating an optionally vectorcoupled complexant of formula I with a diagnostically or therapeuticallyeffective metal ion or metal-containing complex ion.

Metallation may be effected using conventional techniques, eg. reactingthe complexant or a salt thereof in solution with a soluble salt of thedesired metal.

Where, in the compounds of formula I, R¹ groups together withintervening atoms form a cyclic group it is particularly preferred thatthis be a 5 to 8 membered ring containing 0, 1, 2 or 3 heteroatomsselected from N, S and O. More especially it is preferred that one suchheteroatom is provided by a N(R²)_(i) or X group and it is even moreespecially preferred that the R¹ groups are on two carbons adjacent buton different sides of an N(R²)_(i) or X group. Preferably the compoundof formula I will contain zero, one or three such heterocycles,preferably unsaturated and especially preferably aromatic heterocycles,incorporating ring nitrogens oxygens or sulphurs from N(R²)_(i) and Xmoieties. Particularly preferably the resultant heterocycle is anunsaturated N₁, N₂, O₁, N₁O₁ or S₁ heterocycle, preferably a thiophene,pyrrolidine, piperidine, piperazine, morpholine, pyran, pyrrole,imidazole, pyrazine, pyrimidine, imidazolidine, imidazolidinone, furanor pyridine ring. Pyridine, thiophen and furan rings, especiallypyridine rings are especially preferred.

It is also preferred that the two (CR¹ ₂)_(n) groups between theN(R²)_(i) and X moieties should be (CH₂)_(n) or (CR¹ ₂) (CH₂)_(n-1)groups where X is S and where the CR¹ ₂ moieties are attached to theN(R²)_(i) nitrogens. It is further preferred that the (CR¹ ₂) groupsadjacent a (CR¹ ₂) group which is part of a cyclic group themselvesshould be part of a cyclic group or should be CH or CH₂ groups.

It is moreover preferred that the CR¹ ₂ moieties adjacent SR³ groupsshould be CH₂ or CR⁵ ₂, groups (where each R⁵ is independently an alkylgroup, preferably a C₁₋₃ alkyl group), especially preferably CH₂ orC(CH₃)₂ groups. Such CR¹ ₂, moieties are preferably CR⁵ ₂, groups wherethe adjacent (CR¹ ₂)_(n-1)N(R²)_(i) group does not form part of a cyclicgroup.

Where a CR¹ ₂ group is a carbonyl group, this is preferably adjacent aN(R²)_(i) group. Where such a carbonyl group is present it is preferredthat the other CR¹ ₂ group adjacent the N(R²)_(i) group should containan amine or carbonyl function, eg. such a CR¹ ₂ group is a groupCH—CH₂COOH or CH—CH₂CH₂NH₂.

Any cyclic group formed by two CR¹ ₂ groups and intervening atoms may,as indicated above, be optionally substituted, eg. by at least onehydroxy, oxo, halo, alkyl, aryl, amino, CNS, carboxyl or acyl group, eg.by a hydroxy-amino-phenyl group.

Organic groups which are substituents on the compound of formula I willgenerally be C₁₋₂₀ groups, preferably C₁₋₁₀ groups, optionallycontaining one or more, eg. up to six heteroatoms (eg. halo, N, S, P andO atoms). Alkyl, alkenyl, alkynyl and acyl moieties (including alkyleneetc. moieties) will preferably contain up to 6 carbon atoms. Arylmoieties will preferably be phenyl groups or 5 to 7 membered N, S or Oheterocycles. However other hydrophilic substituents, such aspolyalkylene oxides (ie. ((CH₂)_(m)O)_(p) where m is 2 or 3 and p is aninteger of 2 to 500) may be present if desired as biodistributionmodifiers.

Where two NR² ₁ groups are present, it is preferred that in at least oneR² is an amine, carboxyl, or sulfur or phosphorus oxy-acid substitutedC₁₋₆ alkyl group, eg. CH₂CH₂NH₂ or, more preferably, CH₂COOH.

Preferably the compounds of formula I are of formula II

—R³—S—CR⁴ ₂(CH₂)_(n-2)(CR¹ ₂)N(R²)_(i)(CR¹ ₂)(CH₂)_(n-2)(CR¹ ₂)X(CR¹ ₂)(CH₂)_(n-2)(CR¹ ₂)N(R²)_(i)(CR¹ ₂)(CH₂)_(n-2)CR⁴ ₂SR³  (II)

where each CR¹ ₂, which may be the same or different, is CH₂, CH or C,in the later cases being linked to a CR¹ ₂ group adjacent the sameheteroatom to form an optionally substituted saturated or unsaturated 5or 6-membered heterocycle, and each R² where present is H or afunctionalized C₁₋₆ alkyl group (eg. CH₂COOH), preferably one R² beingother than H.

Particularly preferably, each n is 2, X is S or N and 0, 1 or 2 fusedpyridine groups are present in the compounds of the invention. Thus,particularly preferred compounds include those of formulae III to VII:

(where R⁵ is hydrogen or optionally substituted alkyl, aryl, alkaryl oraralkyl;

R⁴ is H or, preferably, CH₃;

R⁶ is H or functionalized alkyl, preferably one being H and the otherbeing CH₂COOH; and X* is a carbon attached heteroaromatic ring, eg. a2,5-thiophene, 2,6-pyridine, 2,5-furan or 2,6-pyrimidine ring,optionally substituted by a R⁵ group)

Direct linkage to a vector group is preferably via a backbone carbon ofa (CR¹ ₂)_(n) moiety or via a ring carbon of a cyclic group formed bytwo (CR¹ ₂) groups and an intervening heteroatom of N(R²)_(i) or X,particularly preferably via a phenyl group attached to such a ring atom.

The thiol protecting group R³ may be any of the known thiol protectinggroups (see for example Greene, “Protective groups in organicsynthesis”, Wiley Interscience, 1981 and McOmie, “Protective groups inorganic chemistry”, Plenum, 1973). Examples of such groups includeoptionally substituted C₁₋₆ alkyl groups, eg. methoxy benzyl (mBz)groups.

The complexants of the invention may be coupled to a vector, a materialwhich will affect the biodistribution of the complexant or itscomplexes, eg. to target it to particular receptors, organs, tissues orbody compartments. Such coupling may be direct or may involve a linker,a bifunctional compound which binds to the complexant and the vector.Examples of suitable vectors include proteins, antibodies, antibodyfragments, oligopeptides, hormones, polyalkylene oxides, andpharmaceuticals. (See for example WO 92/08494).

The compounds of the invention may be prepared by routine organicsynthesis and chelator metallation techniques. Illustrative syntheticschemes are shown below.

The complexant compounds of formula I may be metallated withtherapeutically or diagnostically effective metal ions or complex ions(eg. metal oxide or metal sulphide ions (such as TcO or VO)). Generallyspeaking, preferred metal ions will be radionuclides, paramagnetic ions,fluorescent ions, or heavy metal ions (eg. with atomic number greaterthan 53) or cluster ions.

Examples of appropriate metals include Ag, At, Au, Bi, Cu, Ga, Ho, In,Lu, Pb, Pd, Pm, Pr, Rb, Re, Rh, Sc, Sr, Tc, Tl, Y, and Yb.

Preferred metal radionuclides include ⁹⁰Y, ^(99m)Tc, ¹¹¹In, ⁴⁷Sc, ⁶⁷Ga,⁵¹Cr, ^(177m)Sn, ⁶⁷Cu, ¹⁶⁷Tm, ⁹⁷Ru, ¹⁸⁸Re, ¹⁷⁷Lu, ¹⁹⁹Au, ⁴⁷Sc, ⁶⁷Ga,⁵¹Cr, ^(177m)Sn, ⁶⁷Cu, ¹⁶⁷Tm, ⁹⁷Ru, ¹⁸⁸Re, ¹⁷⁷Lu, ¹⁹⁹Au, ²⁰³Pb and¹⁴¹Ce.

Moreover γ-emitting radionuclides, such as ^(99m)Tc, ¹¹¹In, ⁶⁷Ga and¹⁶⁹Yb have been approved or under investigation for diagnostic imaging,while complexes of β-emitters, such as ⁶⁷CU, ¹¹¹Ag, ¹⁸⁶Re and ⁹⁰Y aremost promising for the applications in tumor therapy. Also γ-emitters(examples are ^(99m)Tc, ¹¹¹In, ⁶⁷Ga and ¹⁶⁹Yb) but also to theβ-emitters (such as ⁶⁷Cu, ¹¹¹Ag, ¹⁸⁶Re, ¹⁸⁸Re and ⁹⁰Y), as well as otherradionuclides of interest (²¹¹At, ²¹²Bi, ¹⁷⁷Lu, ⁸⁶Rb , ¹⁰⁵Rh, ¹⁵³Sm,¹⁹⁸Au, ¹⁴⁹Pm, ⁸⁵Sr, ¹⁴²Pr, 214 Pb, ¹⁰⁹Pd, ¹⁶⁶Ho, ²⁰⁸Tl, and ⁴⁴Sc).Complexes with hard metal ions, such as In³⁺, Ga³⁺, Yb³⁺, and Y³⁺, shallbe stable. In addition, since they contain two or three sulfur atoms,their soft metal (Ag⁺, Cu²⁺, TcO³⁺, and ReO³⁺) complexes should also bestable.

Preferred paramagnetic metal ions include ions of transition andlanthanide metals (eg. metals having atomic numbers of 21-29, 42, 43,44, or 57-71), in particular ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce,Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu, especially of Mn,Cr, Fe, Gd and Dy, more especially Gd.

Preferred fluorescent metal ions include lanthanides, in particular La,Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu. Eu isespecially preferred.

Preferred heavy metal-containing reporters may include atoms of Mo, Bi,Si, and W, and in particular may be polyatomic cluster ions (eg. Bicompounds and W and Mo oxides) as described in WO91/14460, WO92/17215,WO96/40287, and WO96/22914.

All of the publications referred to herein are incorporated herein byreference.

The compounds of the invention may be administered to patients forimaging in amounts sufficient to yield the desired contrast with theparticular imaging technique. Generally dosages of from 0.001 to 5.0mmoles of chelated imaging metal ion per kilogram of patient bodyweightare effective to achieve adequate contrast enhancements. For most MRIapplications preferred dosages of imaging metal ion will be in the rangeof from 0.02 to 1.2 mmoles/kg bodyweight while for X-ray applicationsdosages of from 0.05 to 2.0 mmoles/kg are generally effective to achieveX-ray attenuation. Preferred dosages for most X-ray applications arefrom 0.1 to 1.2 mmoles of the lanthanide or heavy metal compound/kgbodyweight. Where the chelated species is a radionuclide, dosages of0.01 to 100 mCi, preferably 0.1 to 50 mCi will normally be sufficientper 70 kg bodyweight.

The dosage of the compounds of the invention for therapeutic use willdepend upon the condition being treated, but in general will be of theorder of from 1 pmol/kg to 1 mmol/kg bodyweight.

The compounds of the present invention may be formulated withconventional pharmaceutical or veterinary aids, for example emulsifiers,fatty acid esters, gelling agents, stabilizers, antioxidants, osmolalityadjusting agents, buffers, pH adjusting agents, etc., and may be in aform suitable for parenteral or enteral administration, for exampleinjection or infusion or administration directly into a body cavityhaving an external escape duct, for example the gastrointestinal tract,the bladder or the uterus. Thus the compounds of the present inventionmay be in conventional pharmaceutical administration forms such astablets, capsules, powders, solutions, suspensions, dispersions, syrups,suppositories etc. However, solutions, suspensions and dispersions inphysiologically acceptable carrier media, for example water forinjections, will generally be preferred.

The compounds according to the invention may therefore be formulated foradministration using physiologically acceptable carriers or excipientsin a manner fully within the skill of the art. For example, thecompounds, optionally with the addition of pharmaceutically acceptableexcipients, may be suspended or dissolved in an aqueous medium, with theresulting solution or suspension then being sterilized.

For imaging of some portions of the body the most preferred mode foradministering contrast agents is parenteral, e.g., intravenousadministration. Parenterally administrable forms, e.g. intravenoussolutions, should be sterile and free from physiologically unacceptableagents, and should have low osmolality to minimize irritation or otheradverse effects upon administration, and thus the contrast medium shouldpreferably be isotonic or slightly hypertonic. Suitable vehicles includeaqueous vehicles customarily used for administering parenteral solutionssuch as Sodium Chloride Injection, Ringer's Injection, DextroseInjection, Dextrose and Sodium Chloride Injection, Lactated Ringer'sInjection and other solutions such as are described in Remington'sPharmaceutical Sciences, 15th ed., Easton: Mack Publishing Co., pp.1405-1412 and 1461-1487 (1975) and The National Formulary XIV, 14th ed.Washington: American Pharmaceutical Association (1975). The solutionscan contain preservatives, antimicrobial agents, buffers andantioxidants conventionally used for parenteral solutions, excipientsand other additives which are compatible with the chelates and whichwill not interfere with the manufacture, storage or use of products.

The invention is illustrated further by the following non-limitingExamples. Compound numbering is as in the reaction schemes illustratedabove.

EXAMPLE 1

Preparation of 2: To a solution of 76 mg of NaH in 10 mL DMF under N₂,0.27 mL of 4-methoxyl benzyl thiol was added while stirring. Then 0.48 gWIN 63539 solid was added, the mixture was stirred overnight. Themixture was diluted with CHCl₃, washed with H₂O, 10% Na₂CO₃ and brine,dried over Na₂SO₄. It was filtered and 2 was obtained as an off-whitesolid after the solvent was removed by rotary evaporation. The yield is80% and 2 was characterized by TLC and NMR.

EXAMPLE 2

Preparation of 3 and 4: 3 was prepared through a known procedure byreaction of ethyl 2-bromo-2-methyl propionate with HS-MBz in sodiumethoxide/ethanol with yields between 60% to 80%. The crude product wasused in the subsequent in situ generation of 4.

EXAMPLE 3

Preparation of 5: To a solution of 18.2 g of1-Amino-2-methyl-2-propanethiol hydrochloride in 150 mL CH₂Cl₂ and 21 mLtrifloroacetic acid at 0° C., a cold solution of 20.1 g 4-methoxylbenzylchloride in 50 mL CH₂Cl₂ was added dropwise. The mixture was stirred at0° C. for 1 hr and in room temperature for 3 hr. MeOH 30 mL was added tothe mixture to terminate the reaction and all solvents was removed byrotary evaporation. The residue was dissolved in 400 mL CHCl₃, washedwith sat. NaHCO₃ 3×300 mL, 10% Na₂CO₃, H₂O, and brine, dried overNa₂SO₄. It was filtered and 5 was obtained as a colorless oil after thesolvent was removed by rotary evaporation. The yield was 95% and 5 wascharacterized by TLC and NMR.

EXAMPLE 4

Preparation of 6: It was prepared by same procedure as that of 5 usingL-Cysteine ethyl ester hydrochloride. The yield was 95% and 6 wascharacterized by TLC and NMR.

EXAMPLE 5

Preparation of 7: To a solution of 2,6-bis(bromomethyl)pyridine in MeCN,diisopropyl ethylamine and 5 are added, the mixture is heated to refluxfor 3 days and is allowed to cool to room temperature. Extractiontechniques and silica chromatography afford 7.

EXAMPLE 6

Preparation of 8: To a solution of 7 in MeCN, diisopropyl ethylamine andethyl bromoacetate are added, the mixture is heated to reflux overnightand usual extraction techniques and silica chromatography afford 8.

EXAMPLE 7

Preparation of 9: To a solution of 3 (5 g) in CHCl₃, 6.6 mL SOCl₂ wasadded dropwise, then the mixture was refluxed for 3 hr. Solvent wasremoved by rotary evaporation and 50 mL CH₂Cl₂ was added to the residueat 0° C. 3.4 mL Et₃N was added slowly and then a solution of 1.1 g2,2′-bisaminoethyl thioether in 10 mL CH₂Cl₂ was added dropwise. Themixture was allowed to warm to room temperature and then was heated toreflux for 3 hr. It was allowed to cool to room temperature and wastransferred to a separation funnel, washed with sat. NaHCO₃, 10% Na₂CO₃,H₂O, 1N HCl, H₂O, and brine, dried over Na₂SO₄. It was filtered and thecrude product was obtained after the solvent was removed by rotaryevaporation. It was purified by silica chromatography (50%: 50%/ethylacetate : hexane) and 9 was obtained as a colorless oil. The yield was50% and 9 was characterized by TLC and NMR.

EXAMPLE 8

Preparation of 10: It is prepared and isolated in a similar procedure asto that of 9, using diethylene triamine. Usual isolation andpurification procedures afford a pure product.

EXAMPLE 9

Preparation of 11: To a solution of 5.3 g 9 in 40 mL of THF, 40 mL of 1N BH₃.THF was added. The mixture was heated to reflux for 48 hr and wasallowed to cool to room temperature. About 10 mL 6 N NaOH was added todecompose the excess BH₃ and the mixture was refluxed for 30 min. 2 NHCl was added to adjust pH to acidic, and all solvent was removed byrotary evaporation. The residue was dissolved in CHCl3, washed with H₂O,sat. NaHCO₃, H₂O, and brine, dried over Na₂SO₄. It was filtered and thecrude product was purified by silica chromatography (90%: 10%/ethylacetate: MeOH) and 11 was obtained as a colorless oil. The yield was 50%and 11 was characterized by TLC and NMR.

EXAMPLE 10

Preparation of 12: It is prepared and isolated in a similar procedure asto that of 11, using 10 as the starting material. Usual isolation andpurification procedures afford a pure product.

EXAMPLE 11

Preparation of 13: To a solution of 2.9 g 11 in 100 mL MeCN, 0.79 gdiisopropylethyl amine and 0.94 g ethyl bromoacetate were added. Themixture was heated to reflux for 24 hr and was allowed to cool to roomtemperature. Solvent was removed by rotary evaporation and the residuewas dissolved in CHCl₃, washed with H₂O, sat. NaHCO₃, H₂O, and brine,dried over Na₂SO₄. It was filtered and the crude product was purified bysilica chromatography (90%: 10%/ethyl acetate: hexane). It was dissolvedin a mixture of 20 mL THF and 20 mL 5 N NaOH. The mixture was refluxedfor 1 hr and was allowed to cool to room temperature. The pH of thesolution was adjusted to ^(˜)10 with 1 N HCl and it was extracted withCH₂Cl₂. The organic phase was washed with H₂O, 10% Na₂CO₃, H₂O, andbrine, dried over Na₂SO₄. It was filtered and 13 was obtained as a whitesolid. The final yield was ^(˜)40% and 13 was characterized by TLC andNMR.

EXAMPLE 12

Preparation of 14: It is prepared and isolated in a similar procedure asto that of 13, using 12 as the starting material. Usual isolation andpurification procedures afford a pure product.

EXAMPLE 13

Preparation of 15: To a solution of 8.6 g 5 in 150 mL CH₂Cl₂ at 0° C.,5.3 g thioglycolic anhydride solid was added slowly and the mixture wasstirred for 4 hr. It was transferred to a separation funnel, washed withH₂O, 10% Na₂CO₃, H₂O, and brine, dried over Na₂SO₄. It was filtered and15 was obtained as a colorless oil after the solvent was removed byrotary evaporation. The yield was 90% and 15 was characterized by TLCand NMR.

EXAMPLE 14

Preparation of 16: It is prepared and isolated in a similar procedure asto that of 15, using amine-protected iminodiacetic anhydride as thestarting material. Usual isolation and purification procedures afford apure product.

EXAMPLE 15

Preparation of 17: To a solution of 6.5 g 15 in 150 mL CHCl₃, 4.4 gcarbonyldiimidazole solid was added slowly and the mixture was stirredfor 30 min. Then a solution of 5.7 g 6 in 50 mL CHCl₃ was added and themixture was stirred overnight. It was transferred to a separationfunnel, washed with H₂O, 1N HCl, H₂O 10% Na₂CO₃, H₂O, and brine, driedover Na₂SO₄. It was filtered and the crude product was purified bysilica chromatography (90%: 10%/ethyl acetate: hexane). The yield was60% and 17 was characterized by TLC and NMR.

EXAMPLE 16

Preparation of 18: It is prepared and isolated in a similar procedure asto that of 17, using 16 as the starting material. Usual isolation andpurification procedures afford a pure product.

What is claimed is:
 1. A method of generating an image of an animatehuman or non-human animal subject involving administering a contrastagent to said subject and generating by means of a light imagingmodality an image of at least a part of said subject to which saidcontrast agent has distributed, wherein said contrast agent is a metalcomplex of a compound of formula IV

wherein each R³, which may be the same or different, is H or a thiolprotecting group; each R⁴, which may be the same or different, is H orCH₃; R⁵ is hydrogen or optionally substituted alkyl, aryl, alkaryl oraralkyl; and R⁶ is H or CH₂COOH; or a salt thereof.
 2. A method asclaimed in claim 1 wherein the compound of formula IV is metallated withat least one fluorescent metal ion.
 3. A method as claimed in claim 1wherein the compound of formula IV is metallated with at least one metalion selected from the group consisting of the metal ions of La, Ce, Pr,Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.
 4. A method asclaimed in claim 1 wherein the compound of formula IV is coupleddirectly or indirectly to a vector moiety capable of targetingparticular receptors, organs, tissues or body compartments.
 5. A methodas claimed in claim 4 wherein said vector moiety is selected from thegroup consisting of proteins, antibodies, antibody fragments,oligopeptides, hormones and polyalkylene oxides.
 6. A method as claimedin claim 3 wherein the metal ions are of Eu.